BACKGROUND Whole-exome sequencing is a diagnostic approach for the identification of molecular defects in patients with suspected genetic disorders. METHODS We developed technical, bioinformatic, interpretive, and validation pipelines for whole-exome sequencing in a certified clinical laboratory to identify sequence variants underlying disease phenotypes in patients. RESULTS We present data on the first 250 probands for whom referring physicians ordered whole-exome sequencing. Patients presented with a range of phenotypes suggesting potential genetic causes. Approximately 80% were children with neurologic pheno-types. Insurance coverage was similar to that for established genetic tests. We identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients, achieving a 25% molecular diagnostic rate (95% confidence interval, 20 to 31). Among the 62 patients, 33 had autosomal dominant disease, 16 had auto-somal recessive disease, and 9 had X-linked disease. A total of 4 probands received two nonoverlapping molecular diagnoses, which potentially challenged the clinical diagnosis that had been made on the basis of history and physical examination. A total of 83% of the autosomal dominant mutant alleles and 40% of the X-linked mutant alleles occurred de novo. Recurrent clinical phenotypes occurred in patients with mutations that were highly likely to be causative in the same genes and in different genes responsible for genetically heterogeneous disorders. CONCLUSIONS Whole-exome sequencing identified the underlying genetic defect in 25% of consecutive patients referred for evaluation of a possible genetic condition. (Funded by the National Human Genome Research Institute.)

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.

involves karyotyping, whereas in the Netherlands, patients who undergo amniocentesis have a more limited assessment only for trisomies 13, 18, and 21 and the sex chromosomes; thus, the tradeoff is a bit different.In addition, cfDNA is provided as a screening test for trisomies 13, 18, and 21, not just for Down syndrome. However, the performance characteristics of cfDNA for trisomies 13 and 18 are not as favorable as for T21, with a higher rate of falsenegative and false-positive results. In addition, a percentage of patients-somewhere between 1.5% and 8%-fail to obtain a result, usually because of insufficient fetal DNA. Such "low fetal fraction" is associated with obesity, which is a significant problem affecting a high percentage of reproductive-aged women in the United States. It is estimated that 20% to 50% of cfDNA tests fail to provide adequate fetal DNA in obese women. In addition, low fetal fraction is also associated with aneuploidy; therefore, women with cfDNA test failure should be considered high risk and offered follow-up with diagnostic testing (as well as a second attempt at cfDNA). When these potential outcomes are all considered, the performance characteristics of cfDNA versus traditional screening are not as clearly superior.Like the authors of this abstracted paper, several other authors and experts (Prenat Diagn 2013;33 (7):636-642) have suggested a contingent approach, using multiple marker screening as an initial screening tool, and then offering NIPT to intermediate-risk patients and either cfDNA or invasive testing to the highest-risk patients. Before completely changing the current standard of care, we need to understand this tradeoff. These authors consider comparative costs, but do not really provide incremental cost-effectiveness ratios, which are the best way to compare these strategies. While cfDNA is a better test if we are looking at a very precise test for a single disorder, only in patients in whom the test is successful at providing a result current screening may be preferable if we are looking to screen the entire population for a broad range of birth defects. Cost utility analyses, conducted by independent investigators and considering all important outcomes, are clearly needed before our approach completely changes. -MEN)

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.

Early steps for cardiac specification are problematic for the study of mammalian embryos, which has favored using pluripotent cells that recapitulate cardiac myogenesis. Furthermore, circuits governing cardiac specification have relevance to the application of ES cells and other cells for heart repair. In mouse teratocarcinoma cells, canonical Wnts that inhibit heart formation in avian or amphibian embryos and explants activate cardiogenesis, paradoxically. Here, we show that the Wnt/␤-catenin pathway also is essential for cardiac myogenesis to occur in ES cells, acting at a gastrulation-like stage, mediating mesoderm formation and patterning (two prerequisites for cardiac myogenesis itself). Among genes associated temporally with this step was Sox17, encoding an endodermal HMG-box transcription factor. Using lentiviral vectors for RNA interference in differentiating ES cells, an essential role for Sox17 was proven in cardiac muscle cell formation. Sox17 shorthairpin RNA suppresses cardiac myogenesis selectively, acting subsequent to mesoderm formation yet before induction of Mesp1 and Mesp2, a pair of related basic helix-loop-helix transcription factors that together are indispensable for creating heart mesoderm. Sox17 short-hairpin RNA blocks cardiac myogenesis non-cell autonomously and impairs the induction of Hex, a homeodomain transcription factor that is known to be required for the production of endoderm-derived heart-inducing factors.cardiac myogenesis ͉ differentiation ͉ ␤-catenin ͉ heart ͉ Wnt

PurposeWhole exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of whole exome sequencing in adults.MethodsWe performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms.ResultsMolecular diagnoses were reported for 17.5% (85/486) of adults, lower than a primarily pediatric population (25.2%; p=0.0003); the diagnostic rate was higher (23.9%) in those 18–30 years of age compared to patients over 30 years (10.4%; p=0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses.ConclusionEarly WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults.

5q31.3 microdeletion syndrome is characterized by neonatal hypotonia, encephalopathy with or without epilepsy, and severe developmental delay, and the minimal critical deletion interval harbors three genes. We describe 11 individuals with clinical features of 5q31.3 microdeletion syndrome and de novo mutations in PURA, encoding transcriptional activator protein Pur-α, within the critical region. These data implicate causative PURA mutations responsible for the severe neurological phenotypes observed in this syndrome.

Our conditional serum response factor (SRF) knockout, Srf Cko , in the heart-forming region blocked the appearance of rhythmic beating myocytes, one of the earliest cardiac defects caused by the ablation of a cardiac-enriched transcription factor. The appearance of Hand1 and Smyd1, transcription and chromatin remodeling factors; Acta1, Acta2, Myl3, and Myom1, myofibril proteins; and calcium-activated potassium-channel gene activity (KCNMB1), the channel protein, were powerfully attenuated in the Srf CKO mutant hearts. A requisite role for combinatorial cofactor interactions with SRF, as a major determinant for regulating the appearance of organized sarcomeres, was shown by viral rescue of SRF-null ES cells with SRF point mutants that block cofactor interactions. In the absence of SRF genes associated with biomineralization, GATA-6, bone morphogenetic protein 4 (BMP4), and periostin were strongly up-regulated, coinciding with the down regulation of many SRF dependent microRNA, including miR1, which exerted robust silencer activity over the induction of GATA-6 leading to the down regulation of BMP4 and periostin.heart development ͉ microRNA ͉ periostin ͉ cardiogenesis ͉ GATA6